CN102504027B - Preparation of multi-epitope thymidine kinase 1 (TK1) antibody and use of multi-epitope TK1 antibody for early tumor detection and risk early warning in mass physical examination screening - Google Patents
Preparation of multi-epitope thymidine kinase 1 (TK1) antibody and use of multi-epitope TK1 antibody for early tumor detection and risk early warning in mass physical examination screening Download PDFInfo
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Abstract
The invention provides a high-specificity and high-sensitivity coordinated antibody against human thymidine kinase 1 (TK1) prepared from an antigenic determinant consisting of 23 peptides at an N terminal, 20 peptides at a C terminal and 28 peptides at the C terminal of the TK1 monomer from a human hela cell and the use of the detection and diagnosis system of the antibody in tumor diagnosis. The antigenic determinant comprises the following amino acid sequences: the sequence of the 23 peptides (3-25) at the N terminal:CINLPTVLPGSPSKTRGQIQVIL; the sequence of the 20 peptides(206-225) at the C terminal: CPVPGKPGEAVAARKLFAPQ; and the sequence of the 28 peptides (198-225) at the C terminal: AGPDNKENCPVPGKPGEAVAARKLFAPQ. The invention also provides a method for preparing the antibody by using the antigen. An antibody kit provided by the invention has the characteristics of high sensitivity, high specificity, low cost and the like; and the early tumor can be detected and pre-warned in mass physical examination screening by enhanced chemiluminescence dot blot assay, immuno-histochemistry and the detection kit.
Description
Technical field:
The present invention relates to a kind of antibody and application thereof, specifically relate to the preparation of a kind of high specific, highly sensitive multi-epitope TK1 antibody and in crowd's health check-up examination infantile tumour detect and Risk-warning in application.
Background technology:
Cancer is human dead killer, although many image detection methods and treatment means are arranged, such as: chemotherapy, endocrine therapy, radiotherapy and operation etc., although made great progress, but may reach perfect degree far from, with the possibility of recurrence and transfer.Therefore, early stage discovery, particularly discovery and the early stage treatment early than the precancerous lesion phase is the best Gospel that gives Rehabilitation.
Cancer is a kind of chronic disease of abnormal cell proliferation.Cancer cells propagation unusual with out of control is that although abnormal gene expression, therefore not yet canceration is called as the front disease of precancerous condition or cancer owing to the relevant gene of propagation certain has occured in the normal cell or various mutations is caused.Disease refers to that the people who suffers from these diseases will increase greatly than the chance that other people get cancer before the cancer, thereby is also referred to as the front disease of cancer or precancerous condition.And according to the cytopathology concept, a class has the proliferative lesion of cell atypism and prosoplasia, is called precancerous lesion.Cell carcinogenesis is phase in and develops gradually.Attack normal tissue cell to producing cancer from carcinogen, generally need more than 10 years even the longer time, advance rate and course of disease length depend primarily on tumor type and individual difference.Disease progression becomes in this very long process of Cancerous disease before the cancer, also be divided into gently, in, heavily wait different steps.The severe precancerous lesion be before malignant tumour occurs last, also be the most dangerous period.Obviously, preventing and treating precancerous lesion is the most key link of prophylaxis of tumours and tomor rejection, by being paid close attention in the whole world.Yet time and position that precancerous lesion occurs all have uncertainty, and pathology still is in cell levels, not different from sings and symptoms, and existing detection means is difficult in time check to be found.And conventional blood test (formed elements in the blood is that the quality and quantity of these three systems of red corpuscle, white corpuscle and thrombocyte detects and analyzes) and the urine detection used in the existing health check-up can provide the information of the abnormal index such as serum or urine protein or enzyme and relative disease; (B-is super for existing conventional physics image detection method, X-light), the polyp or the polyp that can be observed in the histoorgan increase, the relevant information of tumour or suspected tumor, but these methods still can not be known the polyp that shows in examinee's histoorgan, the abnormality proliferation situation of the tumour cell of tumour or lump, whether very difficult assessment might process be malignant tumour.
What particularly point out is, but up to the present, seldom has believable tumor growth Research of predicting markers to be applied to the health check-up examination.2008, how health care company of U.S. Cigna will now collect for diagnosis and the supervision of cancer with tumor markers (Tumor Markers), propose generally acknowledged criterion and practice guideline, for the blood serum designated object of Tumor-assaciated, assess.Carry out retrospective research for existing many tumor markerses, CEA for example, CA15-3, a series of marks such as CA199 and AFP, because this class mark is not directly related with the propagation of tumour cell, they normally can not come the diagnosis and detection Cancerous disease with the level of independent a kind of tumor markers, this type code thing raises, may indicate cancer, but it is cancer that their existence can not be diagnosed, and these marker detection are just can confirm as cancer in conjunction with other diagnostic modes (for example: biopsy, radiation/imaging).
Later 1950s, the investigator finds thymidine kinase (EC.2.7.1.21 is called for short TK for TK, ATP:thymidine5 '-phosphotransferase), a kind of enzyme of pyrimidine salvage pathway, and catalysis thymidine phosphoric acid turns to thymidylic acid.TK occurs with the form of two kinds of isozyme in people's cell: cytoplasmic thymidine kinase (being called for short TK1) and thymidine kinase,mitochondrial (being called for short TK2).Thymidine kinase 1 (is called for short TK1, be also referred to as cytoplasmic thymidine kinase) be and class mark of closely-related cell cycle of cell cycle regulating, a large amount of fundamental researchs subsequently and experimental oncology have proved that the TK1 expression is closely connected with cell growth state, therefore TK1 can be used as the proliferation activity that a kind of good cell proliferation marker detects proliferative cell, and the propagation degree that therefore will be suitable for malignant tumour detects.
Adopt people TK1 antibody test serum T K1 concentration level more can truly represent the cell that whether has abnormality proliferation in the collective, therefore, in nearly more than 10 years, according to the antibody preparation principle, a kind of TK1 antibody is tried out in clinical practice.A plurality of team have prepared evaluation and the monitoring that different TK1 antibody is used for tumor disease, but are not suitable for conventional crowd's health check-up examination.
On this basis, we are further screening study in 2002, this crucial immune sequence-31 peptide (195-225) of having selected to be exposed to the antigenic determinant C end parts of TK1 protein surface has prepared anti-human TK1-IgY antibody (Chinese invention patent WO204 100760A1, international patent W O 204100760A1.25/11-2004).Employing people TK1C holds the anti-human TK1-IgY antibody of 31 peptides preparation and utilizes detection of plasma and the histogenic immunity lesion detection test kit of its preparation, this antibody will detect activated TK1 albumen and inactive TK1 albumen simultaneously, also can detect the mixture that TK1 protein and other molecule form.Although this serum reagent box-enhancing electrochemiluminescent immunoassay dot blot detection system is tried out in the health check-up examination, sensing range is between 0.3-2pM, but when 0.1-1pM, error is sometimes greater than 30%, employing crowd routine physical examination serum T K1 and cancer patients's serum TK1 carry out the analysis of ROC value, ROC value area under curve is 0.96, specificity is 0.991, maximum likelihood method ratio (+) is 56.04, the specificity of proof antibody is fine, can be used for crowd's health check-up examination, but sensitivity is 0.51%, need further preparation detection sensitivity height, the antibody of specificity and good stability improves detection system, in order to better improve the detection tolerance range in the 0.1-1pM scope.
Summary of the invention:
C holds on the basis of 31 peptides (195-225) as antigen on the TK1 monomer that adopts human cervical carcinoma cell, through for many years practice, filtered out the special epiope of the multi-point that is exposed to the TK1 protein surface, key immune peptide sequence 23 peptides that comprise the N end parts, C holds crucial immune sequence 28 peptides of 20 peptides and C end parts, use the immune sequence of above-mentioned key and do special antigen, adopt the final purifying of hen immunity and combination to prepare anti-human TK1-IgY antibody.Compare with the commercial anti-human TK1-IgG antibody in the existing world, prepared antibody has purity height (＞99%), output height, highly sensitive, high specific and without the non-specific immunity cross reaction, and can more than 1 year and activity stabilized, be convenient to the advantages such as commercial transportation, preservation and operation in+4-8 degree Refrigerator store.Anti-human TK1 multi-epitope Antibody of the present invention and TK1 detection system, the propagation degree that can reflect really cell, thereby can be in situation about detecting early than iconography, the early stage potential abnormal cell proliferation of early warning in the health check-up crowd, the caution examinee may process be the risk of malignant tumour.
Technical scheme provided by the invention comprises:
1, design and screening antigen
The peptide section of N, C end on the protein surface of selecting to expose.
2, Dispersal risk
Utilize crosslinked composition 23 peptides of the immune sequence of above-mentioned key and KLH-KLH, 20 peptides-KLH, the antigen of 28 peptides-KLH go respectively immune hen to make.And utilize the advantage of hen immunity to be: there is the molecular genetic otherness between the IgY of (1) chicken and people's the IgG; (2) TK1 of chicken and people's TK1 has racial diversify; (3) compare with the immune polyclonal antibody for preparing of traditional rabbit, IgY has endogenous molecule homogeneity (only producing one type antibody molecule, i.e. IgY); (4) IgY antibody does not activate people's complement system, thereby partly blocks the activation of non-specific antigen binding site in the human serum; (5) Rheumatoid factors, polyclonal (RF) not with the IgY antibody response.This RF is the main source of non-specific responding in many immunoassay, because the Fc partial reaction of RF and Mammals IgG antibody, can cause the false positive of patient and Healthy Human Serum; (6) present most of patient with breast cancer needs assisting therapy, and many patients accept chemotherapy, for example: He Sai is left alone without help, targeted therapy medicine, monoclonal antibody, yet need later on the hormone antagonist treatment, particularly in the Antybody therapy situation, TK1-IgY antibody demonstrates than single antibody test and has more advantage.
3, antibody purification
Utilize described N to hold 23 peptides, C holds 20 peptides, and C holds 28 peptide sequences crosslinked with KLH respectively, and immune hen extracts yolk liquid respectively, adopts water extraction Dispersal risk solution, and the affinity column with the preparation of the antigenic determinant among the present invention comes antibody purification respectively.
Described purifying comprises the steps:
The first step, just sift out the antibody that immune hen produces and whether have high specific and highly sensitive, namely the yolk liquid of each immune hen separated, obtain the antibody crude product after the extraction of employing water, obtain high specific and the highly sensitive anti-human TK1 antibody of primary dcreening operation through affinitive layer purification.Type according to immunizing antigen is selected affinity column, and mixed crude product liquid is to derive from 23 peptides-KLH, 20 peptides-KLH, and the anti-TK1-IgY antibody of the antigen immune of 28 peptides-KLH, the affinity column preparation is necessary for N and holds 23 peptides, and C holds 20 peptides and C to hold the affinity column of 28 peptides.
Second step, the affinity column of preparation combination immunizing antigen, the proportioning test to 3 kinds of antibody that just sift out make up is characterized in that the selection of combined antigen affinity column proportioning.The preparation affinity column requires the blending ratio according to selected 3 kinds of antibody, calculates corresponding antigenic determinant, i.e. 23 peptides, the per-cent of 20 peptides and 28 peptide amounts.After the test of affinity column chromatography purifying, select best proportioning assembled scheme.
4, the anti-human TK1-IgY antibody of multidigit point antibody combination preparation, unless otherwise indicated, the per-cent among the application is mass percent.
Ratio range: the monospecific antibody of 23 peptide immunity accounts for 25%～45%, and the monospecific antibody of 20 peptide immunity accounts for 20%～40%, and the monospecific antibody of 28 peptide immunity accounts for 20%～55%.
5, identify sensitivity and the specificity of anti-human TK1-IgY antibody
1) confirm that whether Dispersal risk only has specific reaction with people's TK1, the first step adopts the positive and negative cells strain of TK1, and the western method of printing and dyeing is identified the specificity of antibody: utilize lymphoma positive strain CEM TK1
+With negative strain CEM TK1
-Identify.Carry out the western immunity printing and dyeing of TK1 antibody behind the natural gum electrophoresis, CEM TK1 is positive, and strain has an obvious TK1 electrophoresis band, and the negative strain of CEM TK1 does not manifest any band.What confirm antibody is only with the TK1 enzyme immune response to be arranged, and is specific antibody.
2) whether the antibody of confirming preparation only have specific reaction with the serum T K1 of tumour patient, second step adopts the serum specimen of selecting to have confirmed as healthy serum without the disease patient and the positive value of tumour patient, detect with strengthening electrochemiluminescent immunoassay dot blotting method, the serum specimen of the positive value of tumour patient is answered the positive value of showed different, and healthy person should be lower than detection threshold 2pM or not show immune response.Confirmed that antibody and healthy serum without the disease patient without immunological cross-reaction, are specific antibodies, and the intensity reaction of STK1 among the comparison of tumor patients serum, the different antibodies of just sifting out is identified, filter out highly sensitive and specificity good antibody.
3) confirm Dispersal risk whether only with tumour patient in TK1 specific reaction is arranged, from 2) filter out the highly sensitive and good antibody of specificity the step, adopt first visit but do not treat serum and the healthy serum specimen point sample without the disease patient of tumour patient, after carrying out the natural gum electrophoresis, again with the western immunity printing and dyeing of anti-TK1 antibody, the serum specimen of tumour patient shows an obvious TK1 electrophoresis band, and healthy serum specimen without the disease patient does not manifest any band.Confirming that antibody is only with the serum T K1 enzyme of tumour patient immune response to be arranged, is specific antibody.
The present invention utilizes aforesaid combination antibody under 100,000 grades clean environment, and antibody is carried out packing, assembles the TK1 immunity detection reagent, and is combined with chemiluminescent analyzer, is applied to infantile tumour detection and Risk-warning in crowd's health check-up examination.
This invention relates to early prediction, can detect early than iconography in the situation of cancer, for detection of disease before the polytype cancer/tumor disease of hiding in early days/chronic disease and abnormal cell proliferation relevant with tumor disease, the early warning examinee may process be the risk of malignant tumour.STK1 detects and enters combined health checkup services, the risk population that screening STK1 raises, and the disease joint assessment cells in vivo abnormality proliferation situation relevant with following malignant tumour process comprises: regularly dynamic observation of precancerosis disease risk population; In for example organizing/and the severe hyperplasia, the polyp pathology, in/severe ulcer or erosion, viral hepatitis is positive, and for example hepatitis B 135/145, refractory type anaemia disease etc.; The regular monitoring relative disease may fade to cancer disease risks process, innocent tumour for example, in/severe fatty liver, overweight, long-term chronic inflammation relevant with tumor disease etc.; Tumor recovering patient crowd regular monitoring, Risk-warning; Have or not abnormal cell proliferation: 3-6 month STK1 to continue high-level person in the early warning body, but existing Absence of physical signs disease. in the early warning body abnormal cell proliferation is arranged, need and coherent video instrument, tumor markers, serum and urine index associating regular monitoring, the observation of cell abnormality proliferation changes.
MEC detects examinee STK1 level, set up the basal level of the individual STK1 of healthy person, for example without the healthy examinee of tumor disease, need get the first one after another sample in 1 month, analyze the level of STK1,3 middle of the month, carry out duplicate detection 3 times, 3 times detected result is calculated mean value, draw the basal level of the individual STK1 of healthy person, the examinee generally is being lower than 1pM without tumor disease health, during the annual routine physical examination, compares with the basal level of the STK1 value that detects with the STK1 of individuality later on.
Possible process is that the examinee's of cancer disease risks prediction is the basic value and later annual test TK1 value according to the TK1 of individuality, to both STK1 level comparisons.For example, examine now the basis of STK1 value and individual STK1 relatively, if should confirm to have the front disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, but the level of TK1 still is lower than risk threshold value, it is low that the examinee is evaluated as the abnormal cell proliferation level, and process is that the risk of cancer disease is low; If should confirm to have the front disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, the level rising 1-2 of STK1 doubly surpasses 2pM, and the examinee is evaluated as the abnormal cell proliferation level and raises, and the early warning process is that the risk of cancer disease raises.
Adopt this method that the examinee is regularly detected, can monitor the individual several years, so that many decades judges whether the examinee is in cancer disease low risk or high risk condition.Therefore, this invention gives examinee and MEC doctor's early stage information.If STK1 raises, the early warning examinee has the excessive risk of cancer disease process; Might advise the therapeutic intervention scheme of being correlated with giving the killer opportunity that the examinee keeps away from risk and healing to the doctor
With the prior art ratio, advantage of the present invention is as follows:
(1) the prepared antibody of the present invention has that purity height (＞99%), output are high, high specific can reach 0.1pM without non-specific immunity cross reaction and accuracy of detection.Experimenter's working curve represents: ROC area under curve value=0.96 (p＜0.0001), it is 236.49 that maximum likelihood method ratio (+) obviously raises, and specificity is up to 0.997, and sensitivity is up to 0.737.
(2) be applicable to crowd's health check-up examination, can be according to the variation of the different individual instances of examinee and serum T K1 (STK1) level greater than 2pM, indication abnormal cell proliferation degree, information is provided for individual and doctor, strick precaution/therapeutic intervention reasonably keeps healthy, keep away from risk, may greatly improve patient's curative effect.
(3) good stability can be in+4-8 degree Refrigerator store more than 1 year and activity stabilized, the commercial transportation of being more convenient for and preserve and can reduce the implicit costs of reagent.
Description of drawings:
Fig. 1: ABmart antibody analysis test TK1 protein graphical spectrum.
Fig. 2: (a) TK1 antibody kit point sample figure as a result
(b) STK1 dot blotting experimental analysis software test TK1 antibody kit point sample data
(c) STK1 dot blotting experimental analysis software test TK1 antibody kit point sample data and curves figure
Fig. 3: (a) be CEM TK1
+With CEM TK1
-Western part printing and dyeing result.
(b) for identifying the front lymphatic cancer patient for the treatment of, treat front/rear head and neck cancer patient, western part printing and dyeing result of Healthy Human Serum.
Fig. 4: CEM TK1
+, CEM TK1
-, endocervix cancer and colorectal carcinoma TK1 antibody staining result
Fig. 5: the comparison diagram of vitamin H-TK1-IgY antibody (2 footwork) and three-step approach
Fig. 6: the ROC chart that the serum T K1 that MEC provides detects
Fig. 7: the anti-TK1-IgY antibody of Hua Ruitong health and the experiment comparison diagram of the anti-TK1-mAb antibody of commercially available commercialization
Embodiment:
With following embodiment the present invention is described in detail:
Embodiment 1: select the specificity multi-epitope antigen
As shown in Figure 1, with software prediction TK1 no signal peptide, without the cross-film structure.Diagram strand TK1 protein 23 4 amino acids distributions point out that amino acid whose hydrophilic and hydrophobic amino acids distribution is more even.Collection of illustrative plates provides possibility: 1) considers from the antigenicity angle, and better at the polypeptide that the high place of blue curve peak value is selected; 2) according to the distribution of hydrophilic amino acid, select hydrophilic region; 3) select the stronger die aromatischen Aminosaeuren of hydrophobicity shown in the negative value.
Example 2: the antigen of selecting highly sensitive, the anti-human TK1-IgY antibody of high specific multidigit point combination
Select the preparation highly sensitive, the selection of the antigen of the anti-human TK1-IgY antibody of high specific multidigit point combination: have identical epi-position because the partial order of human cervical carcinoma TK1 is shown with other albumen, the antibody that obtains may have cross reaction with other nonspecific proteins, purification difficult, according to announcing known TK1 sequence (protein library), selection is exposed to the special peptide of protein surface, be listed in the table below greater than 85% polypeptide fragment according to the degrees of specificity of the polypeptide that obtains with software prediction, the antigen fragment of the TK1 high specificity of screening, designed TK1N and held 23 peptides, C holds 20 peptides and C to hold 28 peptides as haptens.
Embodiment 3: screening specificity multi-epitope antigen
The selection of specificity multi-epitope antigen: according to known TK1 space structure, selection is exposed to the antigenic determinant on the surface of TK1 protein, according to the strong antigen fragment of example 2 screening TK1 immunitys, the N of screening TK1 holds 23 peptides, C holds 20 peptides, holding 28 peptides with C is the strong antigen fragments of immunity, three sections aminoacid sequence difference following (seeing sequence table): 1) N holds 23 peptides (3-25): CINLPTVLPGSPSKTRGQIQVIL, 2) C holds 20 peptides (206-225): CPVPGKPGEAVAARKLFAPQ, 3) C holds 28 peptides (198-225): AGPDNKENCPVPGKPGEAVAARKLFAPQ.
Embodiment 4: make up anti-human TK1-IgY antibody with the coordination of hen immunity preparation specificity multi-epitope
Use above-mentioned N and hold 23 peptides, C holds 20 peptides, C hold 28 peptide sequences do haptens respectively with the KLH hinge, the preparation immunizing antigen, utilize this antigen to prepare the new combinatorial antibody of anti-human TK-IgY antibody highly sensitive, that specificity is good.The antigen consumption: select 40 of the good healthy Luo Man hens of immunity, consumption is 0.5 milligram, KLH N23 peptide, the KLH-C20 peptide, the KLH-C28 peptide mixed with the Fu Shi Freund's complete adjuvant above-mentioned immunizing antigen with the dissolving of PBS damping fluid again by 1: 1, be expelled to the chest muscle of bird inlay.Through twice to chicken with freund 's incomplete adjuvant and antigen mixed solution booster immunization, all around after every day collect egg, 4 ℃ of lower storages.
Embodiment 5: extract and purifying multidigit point combinatorial antibody
The purifying crude process of antibody is as follows: adopt funnel with mesh with yolk and albumen sepn, complete yolk removes the yolk epidermis with taking the photograph son after cleaning with deionized water, the collection egg yolk liquid.With the distilled water diluting egg yolk liquid of 10 times of volumes, the evenly rear adjust pH of agitating is 5.0, spends the night 4 ℃ of lower placements.Adjust pH to 5.5 after taking out next day is by the secondary ammonium sulfate precipitation method, for the first time according to adding ammonium sulfate solids 351g in the 1000ml solution, add the ratio of 196g ammonium sulfate in every 1000ml the 2nd time, at 10 ℃ of lower frozen centrifugations, rotating speed is 4000 rev/mins, and the time is 24 minutes.Abandoning supernatant, with protein precipitation with the dilution of 1: 4 deionized water and with the NaOH adjusting pH to 8.0 of 0.5mol/L.Adopt the G25 post to remove after the ammonium sulfate affinity column for preparing on the crude product solution of mixing.
1) collection screen is selected the egg of the hen production of good immune effect, and egg yolk is prepared ammoniumsulphate soln, removes after the ammonium sulfate, and crude product TK1 solution mixes upper affinity column.
2) preparation affinity column
Select the type of affinity column antigen: mixed crude product liquid derives from the anti-TK1-IgY antibody of the antigen immune of 23 peptides-KLH+20 peptide-KLH+28 peptide-KLH, affinity column is prepared as 23 peptides+20 peptides+28 peptide mixing affinity columns, the preparation affinity column requires according to selected 3 kinds of antibody blending ratios, calculate corresponding antigen consumption, i.e. 23 peptides, the per-cent of the amount of 20 peptides and 28 peptides is held 23 peptides with calculating good N, and C holds 20 peptides and C to hold the amount interlinkage of 28 peptides to receive affinity column.
3) antibody purification: under 2-8 ℃, will mix crude product liquid and add affinity column, 5 circulations, the speed when every ml affinity column is crossed post the 1st time is 0.2ml/min-0.25ml/min.With affinity column balanced solution flushing foreign protein, through the repeated test more than 3 times, UV stops flushing all less than 0.010 o'clock after the circulation; With the Actisep wash-out antibody of 1.5 times of affinity column volumes, wash-out is removed Actisep with the G25 post again, mixes collecting good antibody, surveys mixed UV value; In time transfer pH to 8.0, add protection reagent, preserve antibody in+4-8 degree refrigerator.
Example 6: detect and identify TK1 test set installed reagents box
Under 100,000 grades clean environment, antibody is carried out packing.Such as Fig. 2 (a), (b), (c) shown in, adopt the enhanced chemiluminescence dot blot that STK1 is carried out stdn and detect and identify, fast and easy, be up to the standards.
Embodiment 7: the sensitivity of screening antibodies and specificity
Identify sensitivity and the specificity of antibody.1) screening sensitivity: select the qualified hen of preliminary assessment, prepare ammoniumsulphate soln with egg yolk, again with the affinity column antibody purification for preparing, obtain purity greater than 99% TK1 antibody, adopt and strengthen electrochemiluminescent immunoassay dot blotting method, with TK1 standard substance (20,6.6,2.2pM) do typical curve, select to have confirmed as the serum of healthy person and the serum specimen of the positive value of the untreated tumour patient of first visit, detect with strengthening electrochemiluminescent immunoassay dot blotting method, the serum specimen of the positive value of tumour patient is answered the positive value of showed different, Healthy People should be lower than detection threshold 2pM or not show immune response, to not responding with people TK1, Healthy Human Serum is had the nonspecific immunity cross reaction, or not with the antibody of Blood of Tumor Patients Chilly TK1 reaction, be defective antibody, deletion.
Identify the specificity of antibody
Cell cultures: adopt conventional cell culture processes, the CEM TK that experiment is used
+, CEM TK
-Cell uses the RPMI-1640 that contains 10% calf serum to cultivate cell count length to 20 * 10 in culturing bottle
6The time, the cell of gathering in the crops is extracted enchylema (lysis buffer: 10mM Tris-HCl, 250mM sucrose, 160mM KCl, 5.6mM NaF, 3.8mM MgCl2,5.0mM ATP, 0.2%NP-40, pH=7.5) with lysis buffer.The positive strain of the CEM TK1 of 20 micrograms/10 microlitres and the negative strain cell extract of CEM TK1 mix with the electrophoretic buffer of equivalent, natural gum electrophoresis (4-10% gradient), and test kit and operation are provided by supplier Invitrogen company.Carry out the western immunity printing and dyeing of TK1 antibody (semi dry electrophoresis jump operation method is provided by Bio-Rad).IgY TK1Ab (100,000X 1mg/ml) adopts human serum sample, IgY TK1Ab concentration (100,000X 1mg/ml).Be the western printing and dyeing of TK antibody results such as Fig. 3 (a), CEM TK1 is positive, and strain has obvious electrophoresis band of TK1, and the negative strain of CEM TK1 does not manifest any band.Verified that antibody only has specific reaction with people TK1.
Fig. 3 (b) is that the western method of printing and dyeing is identified: sample source wherein is the lymphatic cancer patient before 1 example treatment, the 1 front/rear head and neck cancer patient of example treatment, 1 routine Healthy People (without disease), identify through a printing and dyeing/immunostimulant luminescent detection system (ECL), be respectively 44,20,0.5 and 0.1pM.2 microlitre serum dilutions is that 10 microlitres mix with the electrophoretic buffer of equivalent, carries out western immune printing and dyeing of TK1 antibody after crossing natural gum electrophoresis (4-10% gradient).The result shows that the head and neck cancer patient before the treatment before lymphatic cancer patient and the treatment has an obvious electrophoresis band, and the head and neck cancer patient after the treatment and the serum T K1 of Healthy People can observe hardly.Checking antibody and human serum are without the non-specific immunity cross reaction.
Embodiment 8: identify specificity and the sensitivity of TK1 antibody
Paraffin-embedded thick be 4 μ m section preparations, through the dewaxing aquation, the immersion target repair liquid of will cut into slices.According to supplier (
System examination pigment agent box) operation steps that provides is carried out histochemical stain.3%H is immersed in section
2O
2Solution, deactivation endogenous enzyme after 10 minutes was processed 30 minutes with closed reagent, added TK1 antibody, and at room temperature hatched 2 hours; Clean section with PBS solution; Add the EnVisiong mixture, and at room temperature hatched 40 minutes; Diaminobenzidine develops the color and with phenodin slide glass is carried out counterstaining.Positive and the negative human lymphoma cem cell of TK1 adopts the acetone fixation method, and dyeing thereafter and operation steps are ditto described.The TK1 that assesses each section expresses, and the microscopic field by＞10 is observed each tissue slice with the 200-400 magnification, calculates the percentage calculation of pressing at last the positive staining cell with the quantity that contains the TK1 positive cell in＞1000 cells.
Such as Fig. 4, adopt a kind of lymph tumor cell strain: the positive strain of CEM TK1 and the negative strain of CEM TK1 are that standard is identified.Collection of illustrative plates is presented at the positive staining (A) that is in the tenuigenin in the positive strain cell of CEM TK1 in various degree, but dyes in the negative strain of CEM TK1, and reaction (B) is negative.Malignant tumor patient tissue sample-endocervix cancer (C) and colorectal carcinoma (D).Collection of illustrative plates shows the dyeing characteristics: be positive staining in various degree in the tenuigenin in malignant tumour endocervix cancer and colon cancer cell, the part cell also dyes in the showed cell nuclear.Conclusion: TK1 antibody is to have specificity and sensitivity.
Embodiment 9: use TK1-IgY antibody
(1) detection method (being called 3 footworks) during the contracting of nitrocellulose film spot printing and dyeing/immunostimulant luminescent detection system (ECL)
Enhancing based on patent in 2002 is luminous-the immunity point method of printing and dyeing (ECL dot blot Assay).Set up method for quick.Its program is: get early ante cibum for examination person's biological fluid, for example: 1) get fasting blood sample in early morning, centrifugal 4000 turn/8-10 minute separation of serum.With the accurate point sample of the serum sample of 3 microlitres to nitrocellulose filter (HybandTMC, GE, UK), simultaneously, with different concns TK1 (20,6.6,2.2pM) do standard substance point sample (132).Air-dry 30 minutes is 7.5 TBS (20mmol/L Tris, 0.15mol/L NaCl) rinsing (2 minutes/2 times) with pH; 2) add 6% Off fat milk powder with the configuration of TBS damping fluid, sealing is 1 hour under the room temperature, removes closed reagent; 3) add TK1 antibody with the best weaker concn of TBS configuration, immune response 1.5 hours.After reaction is finished, with the rapid rinsing of TBST (adding 0.1% polysorbas20 among the TBST) 2 times, jolt washing 3 times (5 minutes/1 time); 4) add biotinylation the 2nd antibody (by the optimum concn dilution), jolt reaction 40 minutes under the room temperature.After reaction is finished, the same method washing; 5) add avidin-horseradish peroxidase (streptavidin-Horse Radish Peroxidase).After the same method washing, accurately reacted 1 minute with the ECL luminescence reagent, after the drying, this film phonograph seal is in bag film, put into the CCD detecting instrument, the power of this luminous signal will be by CCD (Charged Coupled Device) imaging system analyser lock-on signal and scanning quantitation analysis.According to standard relatively, the concentration of TK1 is value-at-risk greater than normal healthy people level threshold value 2pM.Adopt this typical curve, calculate the change level of examinee TK1, corresponding to the rate of propagation of examinee's tumour cell.Adopt its accuracy of detection of detection method of antibody mediated immunity combination to reach below the 0.1pM.
Embodiment 10: the TK1-IgY antibody of the direct mark of applicating biotin
Such as Fig. 5, the sulfonic group succinimide-direct mark TK1-IgY of LC-vitamin H antibody (2143 test kits
Sulfo-NHS-LC-Biotinylation, Pierce, USA).The sulfonic group succinimide that takes out from-20 degree refrigerators-LC-vitamin H bottle, the method of pointing out is to specifications calculated, the vitamin H reagent of 20 times of mol ratios and the IgG antibody of 1 times of mole, the 2.2 milligrams of vitamin Hs of weighing are dissolved in the ultrapure water without any amine ion of 400 microlitres, the vitamin H reagent of getting 24.1 microlitres joins among the PBS with 975.9 microlitres, disposes 2 milligrams of purifying TK1-IgY antibody.Ice bath was hatched two hours or was at room temperature reacted 30-60 minute.Adopt the molecule number of the vitamin H of HABA Assay certification mark, adopt immediately desalting column to remove unnecessary vitamin H.Adopt experimental example 5 methods, add biotinylation TK1 antibody (by the optimum concn dilution), jolt reaction 1.5 hours under the room temperature, after reaction is finished, again according to experimental example 8 operations, after the method washing, entered for the 5th step.The result is identical with 3 footworks, operates more simple and convenient.
Embodiment 11: identify the sensitivity of antibody
Specificity and sensitivity to the anti-human TK1-IgY antibody of multi-epitope antigen preparation combination are analyzed, the monospecific antibody of the anti-human TK1-IgY antibody of multi-epitope antigen preparation combination (23 peptides+28 peptides+anti-human TK1-IgY antibody of 20 peptide multi-epitope antigens preparation combination was according to 1: 1: 1 ratio) and 28 peptide immunity, the monospecific antibody of the monospecific antibody of 23 peptide immunity and 20 peptide immunity compares.To 11 serum that have been diagnosed as the malignant tumor patient positive, adopt enhancing electrochemiluminescent immunoassay dot blotting method to analyze, the result points out that multi-epitope antigen prepares the single antibody comparison of combinatorial antibody and 28 peptide immunity, in 50% serum specimen, and serum T K1 value rising 20%-50%.The monospecific antibody that multi-epitope antigen prepares combinatorial antibody and 23 or 20 peptide immunity compares, in 95% serum specimen, and serum T K1 value rising 20%-50%.Utilize the serum of 10 Healthy Peoples to analyze, multi-epitope antigen prepares the monospecific antibody of combinatorial antibody and 28 peptide immunity, and the monospecific antibody of the monospecific antibody of 23 peptide immunity and 20 peptide immunity compares, and does not significantly change.Illustrate that multi-epitope antigen prepares combinatorial antibody and improved sensitivity, better distinguish individual cells and whether be in the high or low of abnormality proliferation and increment degree thereof.Adopt ROC (experimenter's working curve, Analysis-It statistics computed in software) to confirm the raising of this antibody sensitivity such as Fig. 6.According to the method described above, the examinee carries out the ROC mapping by data and front malignant tumor patient 721 people of domestic routine clinical treatment that MEC provides the non-malignant tumors disease of participating in routine physical examination to amount to 4103 people, the ROC mapping analysis is pointed out, be 2pM by setting risk threshold value, ROC area under curve value=0.96 (p＜0.0001), it is 236.49 that maximum likelihood method ratio (+) obviously raises, specificity is 0.997, sensitivity is 0.737, the anti-human TK1-IgY antibody of pointing out this specificity T K1 multi-epitope antigen preparation combination has highly sensitive and high specific, is applicable to the health check-up examination.
Example 12: analyze the anti-TK1-IgY antibody of the relatively single epitope antigen preparation of specificity T K1 and sensitivity and the specificity that specificity T K1 multi-epitope antigen prepares the anti-TK1-IgY antibody of combination
The ROC analytical procedure is pointed out, the anti-TK1-IgY antibody of specificity T K1 multi-epitope antigen preparation combination, and maximum likelihood method ratio (+) obviously raises, and the meaningful rising of sensitivity is adopted the CHISQ methods analyst, p=0.028 (586/453,97/50).Form: sensitivity and the specificity of analyzing IgY antibody and the combination IgY antibody that specificity T K1 multi-epitope antigen prepares of the relatively single epitope antigen preparation of specificity T K1
Embodiment 13: each peptide ratio range: 23 peptide 25-30%, and 20 peptide 20-25%, 28 peptide 50-55%, purpose is further to improve detection sensitivity and antibody immunity from interference to be used for early stage Risk Screening
Embodiment 14: the anti-TK1-IgY antibody that compares the preparation of commercial style mono-clonal TK1 antibody and Huarui Tongkang Biotechnology company limited
Adopt the method for quick of nitrocellulose film spot printing and dyeing/enhanced chemiluminescence detection system (ECL).
Its program is: get early and detect for examination person's biological fluid ante cibum.According to standard relatively, the concentration of TK1 is value-at-risk greater than normal healthy people level threshold value 2pM.Adopt this typical curve, the level that will convenient calculate examinee's TK1 in the treatment changes, with the relation corresponding to the rate of propagation of examinee's tumour cell.Adopt the detection method of antibody mediated immunity combination can reach enough low accuracy of detection, particularly can detect the level difference of different healthy individual TK1.Detection limit can reach 0.1pM.Such as Fig. 7 detected result: adopt the anti-TK1-IgY antibody test health of Hua Ruitong health preparation without disease people's serum, its serum T K1 can not detect substantially, but commercial style mono-clonal TK1 antibody (QED Bioscience Inc, San Diego, USA) serum with Healthy People has obvious non-specific immunity cross reaction, and duplicate detection result is similar.Conclusion: commercial style mono-clonal TK1 antibody (QED Bioscience Inc, San Diego, USA) cannot be used for healthy population health check-up examination.
Embodiment 15: distribute according to the serum-concentration to healthy population, 95% crowd is under 1pM, and 5% at 1-2pM, we have set up serum T K1 (hereinafter to be referred as STK1) risk threshold value is 2pM, normal people's group: STK1≤2pM, expression: cell normal growth or low propagation, the canceration risk is low; Risk people's group: STK1＞2pM, the expression abnormal cell proliferation raises, and process is the risk rising of cancer disease in the several years.Risk group and Chinese's Carciuogenesis frequency proportions compare, and the tracking results in 6 years shows: normal group and rising group crowd compare, and process is that the cancer disease risks is elevated to 30 times, and all data communication devices are crossed statistical analysis, and p＜0.005 has statistical significance.
Rising is organized the tracking that 170 people and normal cohort 6,354 people of TK1 have carried out 5-72 month, conclusion to TK1: TK1 rising group process is that the risk of malignant tumour raises 30 times.
STK1 level according to the examinee, set up the basal level of the individual TK1 of healthy person, for example without the healthy examinee of tumor disease, need get for the first time the sample in 1st month, analyze the level of STK1, in 3 middle of the month, carry out duplicate detection 3 times, with 3 times detected result calculating mean value, draw the basal level of healthy individual TK1, healthy examinee generally is lower than 1pM without tumor disease, and during the annual routine physical examination, the STK1 value of utilization detection compares with the basal level of individual TK1 later on.
To may process being that the examinee's of cancer disease risks prediction is the STK1 value of utilizing the basic value of individual STK1 and later on annual test, STK1 level to both compares, comprise: Individual Age, examine now the basis of STK1 value and individual former STK1 relatively, if should confirm to have the front disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, but the level of STK1 still is lower than risk threshold value, and it is low that the examinee is evaluated as the abnormal cell proliferation level, and process is that the risk of cancer disease is low; If should confirm to have the front disease of cancer/innocent tumour disease/chronic disease relevant with tumor disease by individuality, the level rising 1-2 of STK1 doubly surpasses 2pM, and the examinee is evaluated as the abnormal cell proliferation level and raises, and the early warning process is that the risk of cancer disease raises.
Claims (6)
1. the preparation method of a high specific, the anti-human TK1 ?of highly sensitive multi-epitope IgY combinatorial antibody is characterized in that,
Respectively human cervical carcinoma cell TK1 monomer N is held 23 peptides (3 ?25): CINLPTVLPGSPSKTRGQIQVIL, C hold 20 peptides (206 ?225): CPVPGKPGEAVAARKLFAPQ and C hold 28 peptides (198 ?225): AGPDNKENCPVPGKPGEAVAARKL FAPQ and KLH are crosslinked, the difference immune hen, extract yolk liquid, adopt water extraction Dispersal risk solution, hold 23 peptides, C to hold 20 peptides with described N respectively, C holds the affinity column antibody purification of 28 peptides preparation, just sifts out high specific, highly sensitive monospecific antibody;
Described anti-human TK1 ?the IgY combinatorial antibody formed by following antibody by mass percentage: the monospecific antibody of described 23 peptide immunity is 25%～45%, the monospecific antibody of described 20 peptide immunity is 20%～40%, the monospecific antibody of 28 peptide immunity is 20%～55%, and the mass percent sum of described 3 kinds of antibody is 100%.
2. the anti-human TK1 ?of multi-epitope IgY combinatorial antibody that utilizes method gained claimed in claim 1, it is characterized in that described anti-human TK1 ?the IgY combinatorial antibody formed by following antibody by mass percentage: the monospecific antibody of described 23 peptide immunity is 25%～30%, the monospecific antibody of described 20 peptide immunity is 20%～25%, the monospecific antibody of 28 peptide immunity is 50%～55%, and the mass percent sum of described 3 kinds of antibody is 100%.
3. anti-human TK1 ?IgY combinatorial antibody claimed in claim 2, it is characterized in that the TK1 albumen that described combinatorial antibody is selected comprises: have enzymic activity/inactive monomer, 2 or 4 aggressiveness TK1 protein, or albumen composition or the inhibition of TK1 protein and the formation of other molecules.
4. detection method to the anti-human TK1 ?IgY combinatorial antibody described in the claim 2: detection method, the direct mark vitamin H of antibody method when described detection method comprises the contracting of nitrocellulose film spot printing and dyeing/immunostimulant luminescent detection system (ECL).
5. a test kit that utilizes anti-human TK1 ?IgY combinatorial antibody preparation claimed in claim 2 is characterized in that utilizing described anti-human TK1 ?IgY combinatorial antibody to be combined with chemiluminescence detection system and makes.
6. such as right 5 described test kits, it is characterized in that described test kit is used in the detection of crowd's health check-up examination infantile tumour and the Risk-warning.
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| CN104672331B (en) * | 2014-10-14 | 2018-10-09 | 华瑞同康生物技术（深圳）有限公司 | A kind of preparation method of 1 antibody of anti-thymidine kinase and its application in Ureaplasma urealyticum bacterium is proliferated |
| CN106554950A (en) * | 2015-09-24 | 2017-04-05 | 深圳市安群生物工程有限公司 | People's TK1 epitope peptides, antigen, antibody, application and test kit |
| CN106556592B (en) * | 2015-09-24 | 2019-05-21 | 深圳市安群生物工程有限公司 | The chemical luminescence reagent kit and preparation method thereof of quantitative detection people TK1 |
| CN111936522A (en) * | 2018-04-18 | 2020-11-13 | 豪夫迈·罗氏有限公司 | Novel anti-thymidine kinase antibodies |
| CN108960096B (en) * | 2018-06-22 | 2021-08-17 | 深圳市恒天伟焱科技股份有限公司 | Human body identification method based on stereoscopic vision and infrared imaging |
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| CN110872354B (en) * | 2018-09-04 | 2022-11-01 | 华瑞同康生物技术（深圳）有限公司 | Chicken-derived monoclonal antibody and single-chain antibody of mammal cell recombinant anti-human TK1, and preparation method and application thereof |
| CN110878123B (en) * | 2018-09-05 | 2022-08-23 | 华瑞同康生物技术（深圳）有限公司 | anti-TK 1 prokaryotic recombinant single-chain antibody and preparation method thereof |
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